Folate-mediated one-carbon metabolism is a metabolic network of interdependent biosynthetic pathways required for the de novo biosynthesis of purines, thymidylate (“dTMP”), and the remethylation of homocysteine to methionine (FIG. 1) (Barry Shane, Folate Chemistry and Metabolism, in FOLATE IN HEALTH AND DISEASE 1-22 (Lynn B. Bailey ed., Marcel Dekker, Inc. 1995)). Methionine can be converted to S-adenosylmethionine (“AdoMet”), the major one-carbon donor for cellular methylation reactions including the methylation of DNA, RNA, phospholipids, proteins, and small molecules (Barry Shane, Folate Chemistry and Metabolism, in FOLATE IN HEALTH AND DISEASE 1-22 (Lynn B. Bailey ed., Marcel Dekker, Inc. 1995); Fox et al., “Folate-Mediated One-Carbon Metabolism,” Vitam. Harm. 79:1-44 (2008)). Impairments in one-carbon metabolism due to nutrient deficiencies and/or single nucleotide polymorphisms diminish dTMP synthesis, leading to elevated deoxyuridylate (“dUTP”) pools, increased rates of dUTP misincorporation into DNA, and consequently futile cycles of DNA excision repair and chromosomal strand breaks (James et al., “Diet-Induced DNA Damage and Altered Nucleotide Metabolism in Lymphocytes from Methyl-Donor-Deficient Rats,” Carcinogenesis 10(7):1209-1214 (1989); Branda et al., “Folate Deficiency Increases Genetic Damage Caused by Alkylating Agents and Gamma-Irradiation in Chinese Hamster Ovary Cells,” Cancer Res. 53(22):5401-5408 (1993); Duthie et al., “DNA Instability (Strand Breakage, Uracil Misincorporation, and Defective Repair) is Increased by Folic Acid Depletion in Human Lymphocytes in Vitro,” Faseb. J. 12(14):1491-1497 (1998)).
Altered folate metabolism also influences chromatin methylation patterns, including genome-wide CpG hypomethylation and site-specific hypermethylation, and altered histone methylation, which modify gene expression patterns (Wainfan et al., “Methyl Groups in Carcinogenesis: Effects on DNA Methylation and Gene Expression,” Cancer Res. 52(7 Suppl):2071s-2077s (1992); Friso et al., “A Common Mutation in the 5,10-Methylenetetrahydrofolate Reductase Gene Affects Genomic DNA Methylation Through an Interaction with Folate Status,” Proc. Natl. Acad. Sci. U.S.A. 99(8):5606-5611 (2002); Gaudet et al., “Induction of Tumors in Mice by Genomic Hypomethylation,” Science 300(5618):489-492 (2003)).
Therefore, the loss of DNA integrity due to increased genome instability and/or changes in gene expression due to altered genome methylation are candidate causal pathways for folate-mediated pathology. Population and clinical studies have established that impairment of folate-mediated one-carbon metabolism, due to nutritional deficiencies and/or variations in one-carbon metabolism genes increases risk for birth defects including neural tube defects (“NTDs”), chronic diseases including cardiovascular disease, and certain cancers. However, molecular mechanisms have not been established, and biomarkers that predict disease risk have not been established.
For instance, during embryogenesis, the neuroepithelium bends and fuses to form the embryonic neural tube through the process of neurulation. Failure of neurulation results in a spectrum of developmental anomalies collectively referred to as neural tube closure defects. Worldwide prevalence of human NTDs ranges from <1-30 per 10,000 births (INTERNATIONAL CLEARINGHOUSE FOR BIRTH DEFECTS MONITORING SYSTEMS, WORLD ATLAS OF BIRTH DEFECTS (World Health Organization, 2d ed. 2003)). One of the strongest environmental determinants of NTD risk is low maternal folate status (Kirke et al., “Maternal Plasma Folate and Vitamin B12 are Independent Risk Factors for Neural Tube Defects,” Q. J. Med. 1993; 86:703-8 (1993)), which interacts with specific gene variants to confer NTD risk (Relton et al., “Low Erythrocyte Folate Status and Polymorphic Variation in Folate-Related Genes are Associated with Risk of Neural Tube Defect Pregnancy,” Mol. Genet. Metab. 81:273-81 (2004); Christensen et al., “Genetic Polymorphisms in Methylenetetrahydrofolate Reductase and Methionine Synthase, Folate Levels in Red Blood Cells, and Risk of Neural Tube Defects,” Am. J. Med. Genet. 84:151 7 (1999)).
Low maternal folate status is one of the strongest environmental determinants of neural tube defect risk (Kirke et al., “Maternal Plasma Folate and Vitamin B12 are Independent Risk Factors for Neural Tube Defects,” Q. J. Med. 86(11):703-708 (1993)) and interacts with specific gene variants to confer NTD risk (Relton et al., “Low Erythrocyte Folate Status and Polymorphic Variation in Folate-Related Genes are Associated with Risk of Neural Tube Defect Pregnancy,” Mol. Genet. Metab. 81(4):273-281 (2004); Christensen et al., “Genetic Polymorphisms in Methylenetetrahydrofolate Reductase and Methionine Synthase, Folate Levels in Red Blood Cells, and Risk of Neural Tube Defects,” Am. J. Med. Genet. 84(2):151-157 (1999)). Periconceptional folic acid intake at a level of 400 μg per day is recommended to all women of childbearing age to reduce the occurrence of NTDs (CDC, “Recommendations for the Use of Folic Acid to Reduce the Number of Cases of Spina Bifida and Other Neural Tube Defects,” MMWR Recomm. Rep. 41(1-7) (1992)). Furthermore, food folic acid fortification has been introduced in the US, Canada, and Chile (Honein et al., “Impact of Folic Acid Fortification of the US Food Supply on the Occurrence of Neural Tube Defects,” JAMA 285(23):2981-2986 (2001)) and has significantly reduced rates of neural tube defects (Czeizel et al., “Prevention of the First Occurrence of Neural-Tube Defects by Periconceptional Vitamin Supplementation,” N. Engl. J. Med. 327(26):1832-1835 (1992); Medical Research Council “Prevention of Neural Tube Defects: Results of the Medical Research Council Vitamin Study. MRC Vitamin Study Research Group,” Lancet 338:131-7 (1991)). However, the folate-dependent metabolic pathway(s) that affect neural tube closure are unknown. It is estimated that only 70% of NTDs are responsive to dietary folic acid (Berry et al., “Prevention of Neural-Tube Defects with Folic Acid in China. China-U.S. Collaborative Project for Neural Tube Defect Prevention,” N. Engl. J. Med. 341(20):1485-1490 (1999)). Other risk factors for NTD affected pregnancies include environmental and food-based toxins (Bhatt, “Environmental Influence on Reproductive Health,” Intl. J. Gynecology & Obstetrics 70(1):69-75 (2000); Hutz et al., “Environmental Toxicants and Effects on Female Reproductive Function,” Trends Rep. Biol. 2:1-11 (2006)), and obesity (Leddy et al., “The Impact of Maternal Obesity on Maternal and Fetal Health,” Rev. Obstetrics & Gynecology 1(4):170-178 (2008)) and maternal diabetes (Hendricks et al., “Effects of Hyperinsulinemia and Obesity on Risk of Neural Tube Defects Among Mexican Americans,” Epidemiology 12(6):630-635 (2001)). It is not known what fraction of the 30% of NTDs that are not folic acid responsive result from impairments in folate-mediated one-carbon metabolism, including metabolic disruptions resulting from vitamin B12 deficiency. Although it has been appreciated that genetic variants interact with folate status to influence NTD risk (Wlodarczyk et al., “Spontaneous Neural Tube Defects in Splotch Mice Supplemented with Selected Micronutrients,” Toxicol. Appl. Pharmacol. (2005)), the vast majority of the genetic risk has yet to be identified (Beaudin et al., “Insights into Metabolic Mechanisms Underlying Folate-Responsive Neural Tube Defects: A Minireview,” Birth Defects Res. A Clin. Mol. Teratol. 85(4):274-284 (2009)).
As noted above, the causal metabolic pathways underlying folic acid-responsive NTDs have not been established. Further, folic acid supplementation has been linked to cancer prevalence (Ebbing et al., “Cancer Incidence and Mortality After Treatment With Folic Acid and Vitamin B12,” JAMA 302(19): 2119-2126 (2009)). Thus, there is a great need for understanding the mechanisms underlying occurrence and recurrence of folate-deficiency related birth defects, as well as alternatives to folic acid supplementation.
The interactions among nutrients and genetic factors also play an important role in the development of numerous cancers including colorectal cancer (“CRC”). A strong, inverse association of folate status and CRC has been demonstrated; individuals with lowest dietary folate intake show a 40% to 60% increase in CRC risk when compared with individuals with highest folate intake (Giovannucci et al., “Folate, Methionine, and Alcohol intake and Risk of Colorectal Adenoma,” J. Natl. Cancer Inst. 85:875-84 (1993); Ma et al., “Methylenetetrahydrofolate Reductase Polymorphism, Dietary Interactions, and Risk of Colorectal Cancer,” Cancer Res. 57:1098-102 (1997); Kim et al., “Folate Intake and the Risk of Colorectal Cancer in a Korean Population,” Eur. J. Clin. Nutr. 63:1057-64 (2009)). Genetic variation that alters folate metabolism and utilization also influences cancer risk (Ma et al., “Methylenetetrahydrofolate Reductase Polymorphism, Dietary Interactions, and Risk of Colorectal Cancer,” Cancer Res. 57:1098-102 (1997)). The mechanism by which folate metabolism alters CRC risk is not known, which has led to concerns regarding the potential impact of elevated dietary folate intake and folate fortification initiatives on CRC incidence (Cole et al., “Folic Acid for the Prevention of Colorectal Adenomas: A Randomized Clinical Trial,” JAMA 297:2351-9 (2007); Logan et al., “Aspirin and Folic Acid for the Prevention of Recurrent Colorectal Adenomas,” Gastroenterology 134:29-38 (2008)).
The present invention is directed to overcoming these and other deficiencies in the art.